Amoebicidal activity of cationic carbosilane dendrons derived with 4-phenylbutyric acid against Acanthamoeba griffini and Acanthamoeba polyphaga trophozoites and cysts

Amoebae from the genus Acanthamoeba are important pathogens responsible for severe illnesses in humans such as Acanthamoeba keratitis and granulomatous amoebic encephalitis. In the last few decades, AK diagnoses have steadily increased. Most patients suffering from AK were contact lens users and the infection was related to poor hygiene. However, therapy is not yet well established, and treatments may last for several months due to resistance. Moreover, these treatments have been described to generate cytotoxicity. Therefore, there is an urgent need to develop new therapeutic strategies against AK. In this study, the amoebicidal activity of different generation cationic carbosilane dendrons derived with 4-phenylbutyric acid was demonstrated against Acanthamoeba polyphaga and Acanthamoeba griffini trophozoites and cysts. In addition, the combination of chlorhexidine digluconate and the most effective dendron (ArCO2G2(SNMe3I)4) showed an in vitro effect against Acanthamoeba trophozoites and cysts, reducing the minimal trophozoite amoebicidal concentration as well as concentrations with cysticidal activity.

Secondly, the dendrons were twofold serially diluted from stock solutions to reach final concentrations ranging from 2 to 512 mg/L and 100 μL of each concentration were added to each well. Then, 100 μL of the adjusted trophozoite solution (2 × PYG + B and 2 × CERVA for A. polyphaga 2961 and A. griffini MYP2004, respectively) were also added. Plates were incubated at 32 °C for A. polyphaga 2961 and 37 °C for A. griffini MYP2004. Manual counting using the 0.2% Congo Red exclusion assay was performed at 24 and 48 h of treatment and percent viability was defined as: % viability = (mean treated/mean control) × 100. The minimum trophozoite amoebicidal concentration (MTAC) was defined as the lowest concentration of test solution that produced a complete reduction in trophozoite viability 18 .
Each drug concentration was tested in triplicate and in at least two independent experiments. A control well containing the amebae with no treatment and control wells of medium were included. All microtiter plates were sealed with Parafilm ® . These procedures apply to all tests carried out in this study.
Cysticidal activity assays. Cysts were obtained from ameba cultures in logarithmic phase under optimal growing conditions. The medium was replaced with Neff 's encystment medium and flasks were agitated for 9 days at room temperature 17 . A. polyphaga 2961 and A. griffini MYP2004 cyst assays were run in 96-well microtiter plates (Deltalab).
The inoculum was adjusted in Neff 's encystment medium, and 10,000 cysts were inoculated per well for both strains. The dendrons were twofold serially diluted to final concentrations ranging from 2 to 512 mg/L; 100 μL of each concentration were mixed with 100 μL of adjusted cyst solution. The dendron solutions were removed after 24 and 48 h of treatment, then each well was washed with 1 × PBS (10x, Sigma Aldrich, St. Louis, MO, USA) and culture medium was added (1 × PYG + B for A. polyphaga 2961 and 1 × CERVA A. griffini MYP2004). Plates were incubated at optimal growing conditions (32 °C for A. polyphaga 2961 and 37 °C for A. griffini MYP2004). Wells were observed three times per week with an inverted microscope (Motic AE21) for 21 days to visualize excystment and determine the minimum cysticidal concentration (MCC), defined as the lowest concentration that completely inhibits excystment and trophozoite growth 18 . Combined treatment against trophozoites. To perform these assays, the procedure was the same as for the amoebicidal activity assays described above. For this purpose, the checkerboard method was used 19 . The most effective dendron was tested in combination with CLX in a final volume of 200 μL (1:1 ratio). Required concentrations were achieved by serial dilution. Dendron concentrations ranged from 0.5 to 16 mg/L (< MTAC), while CLX concentrations ranged from 0.5 to 4 mg/L (< MTAC). Manual counting was performed after 24 and 48 h of incubation under optimal growing conditions to determine trophozoite viability compared to the untreated control.
Combined treatment against cysts. Experiments were prepared as explained above for the cysticidal activity assays. Briefly, 100 μL of adjusted cyst inoculum was added to each microtiter plate well. The combination of the most effective dendron in concentrations ranging from 2 to 64 mg/L (MCC) and CLX, in concentrations from 1 to 8 mg/L, were added to each well (50 μL of each compound). After 24 and 48 h of treatment, the compounds were discarded. Wells were then washed twice with 1 × PBS (10x, Sigma Aldrich, St. Louis, MO, USA) and fresh medium was added. Finally, plates were incubated under optimal growing conditions. They were observed three times per week using an inverted microscope (Motic AE21) for 21 days to determine excystment. To determine synergy, the FICI was calculated as previously described.

Cytotoxicity assay in HeLa cells. HeLa cells were cultured in Dulbecco's Modified Eagle Medium
(DMEM) (Gibco) supplemented with 10% fetal bovine serum and 1% of an antibiotic mixture. The inoculum was adjusted to 1 × 10 4 cells per well in a 24-well microtiter plate. Cells were grown for 3-4 days at 37 °C with 5% CO 2 to reach confluence 17 . Then, the medium was discarded and 400 μL of the most effective dendron and CLX concentrations diluted in DMEM medium were added.
Viability was calculated as (absorbance mean of treated/absorbance mean of non-treated) × 100. Values higher than 90% viability were considered non-cytotoxic, while values between 75 and 90% were considered low cytotoxicity. If viability descended to the range of 60-75%, it was deemed a moderate cytotoxic level. High cytotoxicity was established when viability values were lower than 60% 21 .
Scanning electron microscopy (SEM). SEM study was performed to evaluate the impact of the most effective dendron, CLX and combinations of these as previously described by our group 16 . To perform these studies, 200 μL of adjusted trophozoite suspension were placed on a glass coverslip. After 1 h of incubation under
Cysticidal activity assays. The MMC of the dendritic compounds on the excystment of cysts was determined by observation under the microscope 18 . Only ArCO 2 G 2 (SNMe 3 I) 4 (2) demonstrated a cysticidal activity  (Table 3). For A. griffini MYP2004, the MCC was 64 mg/L, while for A. polyphaga 2961 it was higher, 128 mg/L (Table 3). There was no change between both times of incubation.
Combined treatment against trophozoites. The effective concentrations used in the combination treatments were lower than the effective concentrations for each compound tested individually. Moreover, when the FICI was calculated, a synergistic effect was found when 16 mg/L ArCO 2 G 2 (SNMe 3 I) 4 (2)/1 mg/L CLX were combined against A. griffini trophozoites, and 16 mg/L ArCO 2 G 2 (SNMe 3 I) 4 (2)/0.5 mg/L CLX against A. polyphaga trophozoites. Therefore, the ArCO 2 G 2 (SNMe 3 I) 4 (2) effective concentration was reduced from 64 to 16 mg/L and the CLX effective concentration was reduced from 2-4 mg/L to 0.5-1 mg/L (non-cytotoxic concentrations). Under these conditions, the combined therapy improved compound efficacy and reduced the effective concentrations and thus, the cytotoxicity of both compounds. Nevertheless, some of the other effective combinations had additive or indifferent effects (Tables 4 and 5, Fig. 3).
Combined treatment against cysts. As described in the combination assay against trophozoites, effective treatment concentrations against cysts were lower in combination than for each of the drugs individually. FICI values showed a synergistic effect for the highest ArCO 2 G 2 (SNMe 3 I) 4 (2) concentrations tested in combination. For example, synergy was found when 16 mg/L ArCO 2 G 2 (SNMe 3 I) 4 (2)/2 mg/L CLX were combined against A. griffini cysts, and 32 mg/L ArCO 2 G 2 (SNMe 3 I) 4 (2)/2 mg/L CLX against A. polyphaga cysts. Therefore, the ArCO 2 G 2 (SNMe 3 I) 4 (2) effective concentration was reduced from 64-128 to 16-32 mg/L and the CLX effective concentration was reduced from 8 to 1-2 mg/L. Under these conditions, the combined therapy improved compound efficacy and reduced the effective concentrations against cysts and, in consequence, the cytotoxicity of both compounds. However, the primary outcome was an additive effect, once again (Tables 6 and 7).

Alone
In combination  www.nature.com/scientificreports/ Table 6. FICI in cysts and its interpretation in A. griffini MYP2004. MCC (mg/L) for ArCO 2 G 2 (SNMe 3 I) 4 (2) and CLX alone or in combination after 24 and 48 h of treatment.   The effective treatment concentrations achieved in different combinations against cysts were higher than the ones used against trophozoites that showed high cytotoxicity. Therefore, further cytotoxicity was not evaluated, as it was assumed to be greater than in those treatment combinations previously studied, i.e., more cytotoxic than the combinations of concentrations that were effective against trophozoites.
As observed in the images, CLX completely disintegrates the Acanthamoeba membrane, causing the release of cytoplasmic constituents (Figs. 5D,E, 6D,E), reducing the number of acanthopodia and inducing a rounded shape in trophozoites (Figs. 5D,E, 6D,E). These observations may suggest that dendron ArCO 2 G 2 (SNMe 3 I) 4 (2) caused the appearance of holes, possibly due the interaction with the negative charges on the amoeba surface www.nature.com/scientificreports/ and, in consequence, it may produces the disruption of the plasma membrane (Fig. 6B (arrow)). In addition, after treatments with drug combinations, no other changes were noticed apart from the ones aforementioned for each of the treatments as monotherapies (Figs. 5F,G, 6F,G). Alterations were more evident after 48 h of treatment.

Discussion
AK is a rare, sight-threatening disease, although the severity of its outcomes together with the lack of effective treatments has made the development of new therapeutic agents an urgent line of research 3,24 .
On the one hand, CLX is a biguanide considered a standard treatment drug in AK cases. It can be used in combination with a diamidine or as a monotherapy 24,25 . However, it is highly cytotoxic at effective concentrations 26,27 . To solve this problem, combination strategies have been proposed 15 . On the other hand, dendritic compounds have shown effectiveness in previous studies not only against bacteria and viruses, but also against amoebae 17,[28][29][30] . Moreover, these compounds are water-soluble, and their structure facilitates a directed design, characteristics that make them suitable therapeutic agents 10 . Their cationic structure, like CLX, may explain their biocidal www.nature.com/scientificreports/ activity, even though their mode of action has not yet been completely elucidated 31 . The three dendrons evaluated, ArCO 2 G 1 (SNMe 3 I) 2 (1), ArCO 2 G 2 (SNMe 3 I) 4 (2) and ArCO 2 G 3 (SNMe 3 I) 8 (3), were effective against trophozoites at different concentrations. In addition, the second generation dendron, ArCO 2 G 2 (SNMe 3 I) 4 (2), with four positive charges on the periferial groups and PBA at the focal point showed cysticidal activity. Cysts have the ability to resist treatment by different compounds because they form a protective wall, composed mainly of cellulose (endocyst), proteins and polysaccharides (exocyst) 32 . Nevertheless, A. griffini MYP2004 cysts seemed to be more sensitive than A. polyphaga 2961 cysts to treatment with ArCO 2 G 2 (SNMe 3 I) 4 (2). Although this result may seem to be contradicted by the data shown in literature, the dendron could act differentially on each cyst wall depending on its exact composition 3 . Previous studies described differences in cystic proteins within isolates, so these data may also indicate protein variability between the Acanthamoeba strains used in our study and other amoebic strains 33 . CLX, which caused the alterations observed by SEM after treatments, is a cationic molecule that interacts with the negatively charged plasma membrane, thus provoking membrane destabilization, pore formation, cytoplasmic content leakage and cell death 14,34 . Dendrons are also cationic molecules, thus their mode of action might be similar and alterations may be due to positive charges that interact with and disrupt cell structures 31 . In addition, dendrons have an aromatic ring at their focal point (growing dendron point where peripherial groups emerge) that could improve their antibacterial activity 35 .
In previous studies, amoebicidal activity against A. polyphaga 2961 trophozoites and cysts shown by dendritic wedge topology systems with isobutyric acid at the focal point (IC 50 at 24 h treatment: ArCO 2 G2(SNMe 3 I)4 for trophozoites 8.24 mg/L and for cysts 128 mg/L) was greater than that shown by spherical topology systems containing a polyphenoxy nucleus (IC 50 at 24 h treatment: G1O 3 (SNMe 3 I) 6 for trophozoites 16.9 mg/L and for cysts > 512 mg/L) or a silicon atom nucleus (IC 50 24 h G1O 3 (SNMe 3 I) 6, 430.1 mg/L for trophozoites and > 512 mg/L for cysts) 36 . In view of these results and considering that they all have a similar number of positive charges in their structure, we suggest that the presence of the aromatic ring modulates the activity of the dendritic system, and the topology of the system also seems to have a clear influence on the activity. The dendritic wedge presents a more open structure that could provide the aromatic ring with the possibility for interacting with the amoeba membrane.
The FICI index was calculated in the combination assays 15,37 . The main goal was to find a combination that could reduce effective doses to less cytotoxic ones. To this end, the most effective dendron ArCO 2 G 2 (SNMe 3 I) 4 (2) was tested in combination with CLX. The results showed a synergistic effect at highly cytotoxic concentrations, although other combinations with additive effects achieved a reduction in the effective concentration and had moderate cytotoxicity. For example, certain combinations were able to reduce A. griffini MYP2004 viability to only 3% and completely eliminate A. polyphaga 2961 trophozoites (0% viability). Consequently, we highlight those combinations which achieved a reduction in effective concentrations of CLX with an increase in amoebicidal activity and a significant reduction in cell cytotoxicity. We also propose that the use of these combinations in prevention and treatment strategies could be studied in in vivo models. Additionally, combined therapy has been shown to prevent the development of new resistant strains, thus combination strategies may have extra value in the fight against AK resistance 15 . In summary, our data support the suitability of these promising dendrons alone and in combination for treating AK.

Conclusions
The cationic dendrons with isobutyric acid at the focal point evaluated in this study showed amoebicidal activity against trophozoites. Furthermore, the second generation dendron (ArCO 2 G2(SNMe 3 I) 4 ) (2) with four positive charges on the surface had cysticidal activity and a synergistic effect when combined with CLX, a standard treatment drug. Besides, the combinations with a drastic reduction in trophozoite viability, even to 0%, showed only moderate cytotoxicity. Additionally, the ArCO 2 G2(SNMe 3 I) 4 (2) dendron and CLX modes of action showed similarities as both were capable of disrupting trophozoite membranes.

Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.